N-Substituted l-Iminosugars for the Treatment of Sanfilippo Type B Syndrome

Sanfilippo syndrome comprises a group of four genetic diseases due to the lack or decreased activity of enzymes involved in heparan sulfate (HS) catabolism. HS accumulation in lysosomes and other cellular compartments results in tissue and organ dysfunctions, leading to a wide range of clinical symptoms including severe neurodegeneration. To date, no approved treatments for Sanfilippo disease exist. Here, we report the ability of N-substituted l-iminosugars to significantly reduce substrate storage and lysosomal dysfunctions in Sanfilippo fibroblasts and in a neuronal cellular model of Sanfilippo B subtype. Particularly, we found that they increase the levels of defective α-N-acetylglucosaminidase and correct its proper sorting toward the lysosomal compartment. Furthermore, l-iminosugars reduce HS accumulation by downregulating protein levels of exostosin glycosyltransferases. These results highlight an interesting pharmacological potential of these glycomimetics in Sanfilippo syndrome, paving the way for the development of novel therapeutic approaches for the treatment of such incurable disease.


■ INTRODUCTION
Sanfilippo syndrome, or mucopolysaccharidosis (MPS) type III, is a lysosomal storage disease (LSD) consisting of four disease subtypes, namely Sanfilippo A, B, C, and D, all caused by deficiencies of enzymes involved in the catabolism of the glycosaminoglycan (GAG) heparan sulfate (HS). 1 Defective enzymes are N-sulfoglucosamine sulfohydrolase (SGSH) (EC 3.10.1.1) for Sanfilippo A, N-acetyl-α-D-glucosaminidase (NAGLU) (EC 3.2.1.50) for Sanfilippo B, acetyl-CoA:αglucosaminide N-acetyltransferase (HGSNAT) (EC 2.3.1.78) for Sanfilippo C, and N-acetylglucosamine-6-sulfate sulfatase (GNS) (EC 3.1.6.14) for Sanfilippo D. Although all Sanfilippo subtypes are rare diseases, Sanfilippo A and B are more common than C and D with the incidence of 0.29−1.89 and 0.42−0.72 per 100,000 births, respectively. 2,3 In all Sanfilippo subtypes, as a result of enzymatic defects, undegraded HS accumulates in cellular substructures, like lysosomes and cell membrane, leading to a plethora of clinical manifestations, which involve various organs and tissues including skeletal muscles, heart, lungs, and especially the central nervous system (CNS). 4 Indeed, the clinical course of patients affected by Sanfilippo disease is characterized by progressive CNS degeneration, leading to cognitive decline and autism spectrum disorders. 4 Several efforts have been devoted to the identification of novel treatments for the Sanfilippo syndrome. To date, the main strategies involve (i) substrate reduction therapy (SRT), based on the administration of drugs that inhibit HS synthesis; (ii) enzyme replacement therapy (ERT), aiming to deliver the missing enzyme to target tissues; (iii) gene therapy (GT) that leverages viral vectors�such as adeno-associated vectors (AAVs) or lentiviral vectors (LVs)�in order to transfer the correct enzyme-coding genes, and (iv) hematopoietic stem cell transplantation (HSCT), which supplies the enzyme secreted by healthy donor cells to recipient patients. 5 Although all the aforementioned therapies have demonstrated promising results in preclinical studies, 6−9 clinical ones highlighted no-to-low effect on patients, especially for CNS impairment. 10−12 Thus, the identification of novel therapeutic approaches for this intractable disease is still highly demanding.
Over the last few decades, iminosugars, glycomimetics with an amino function replacing the endocyclic oxygen of natural carbohydrates, exhibited a notable pharmacological potential in the LSD treatment stewardship, thanks to their ability to interact with carbohydrate-processing enzymes and alter their properties. 13 −16 In particular, some iminosugars have found application in the treatment of LSDs either for their ability to inhibit substrate synthesis (SRT) 17,18 and consequent lysosomal accumulation or to bind, reversibly and at sub-inhibitory concentrations, mutated lysosomal enzymes, thus enhancing or restoring their function (pharmacological chaperone therapy�PCT). 19,20 To date, two iminosugars are commercially available for the treatment of LSDs: ZAVESCA (miglustat, also known as D-NBDNJ, N-butyl-D-deoxynojirimycin, compound 2) (Figure 1), licensed within the SRT for the treatment of type I Gaucher's disease 21,22 and Niemann−Pick type C disease, 23 and Galafold (migalastat, also known as DGJ, 1deoxygalactonojirimycin), at present the only approved pharmacological chaperone for Fabry disease. 24,25 In addition, many other iminosugars have been evaluated for their use as drug candidates in different LSDs, including MPSs. 26,27 In this field, the activity of some iminosugar derivatives acting as pharmacological chaperones for the treatment of MPS II, III, and IV was evaluated. 28−31 Furthermore, an interesting application of iminosugars in MPSs involves the assumption that inhibition of ganglioside secondary storage can represent a therapeutic strategy for patients with neurological involvement. On these bases, miglustat was evaluated as a substrate-reducing agent for Sanfilippo diseases due to its ability to interfere with glycosphingolipid metabolism. 32 However, despite the promising results obtained in preclinical studies, 33 no beneficial effects were observed in Sanfilippo patients treated with miglustat. 34 Overall, these findings suggest that iminosugars represent attractive drug candidates for the treatment of Sanfilippo disease and therefore further efforts should be devoted to identify novel iminosugars effective in reducing HS and lysosomal accumulation in Sanfilippo patients. In the frame of our previous studies on the role of chirality in the pharmacological activity of iminosugars and other bioactive compounds, 35−38 we recently highlighted the promising potential of unnatural L-glucoconfigured iminosugars for the treatment of rare diseases. Particularly, L-NBDNJ (ent-2, Figure 1), the synthetic enantiomer of D-NBDNJ (2), showed an interesting potential for the treatment of Pompe lysosomal disease, while not acting as an inhibitor of the most common glycosidases, differently from its enantiomer. 39 Even more interesting results have been obtained by us when L-iminosugars were evaluated in cystic fibrosis (CF). 40 Indeed, ent-2 and its N-substituted congeners exhibited strong anti-inflammatory and antibacterial properties in vitro and in vivo, pointing out the potential use of these compounds for the treatment of CF lung disease. 40,41 Based on the established therapeutic activity of D-iminosugars in the treatment of LSDs, as well as on the promising biological properties exhibited by the unnatural L-iminosugars, 42−44 the pharmacological potential of seven L-iminosugars was herein evaluated in Sanfilippo B disease. L-Deoxynojirimycin (L-DNJ, ent-1), its N-alkyl derivatives (N-butyl L-DNJ, L-NBDNJ, ent-2; N-nonyl L-DNJ, l-NNDNJ, ent- 3), and N-alkoxyalkyl derivatives (N-hexyloxypentyl L-DNJ, L-HPDNJ, ent-4; N-nonyloxypentyl L-DNJ, L-NPDNJ, ent-5; N-adamantanemethoxypentyl L-DNJ, L-AMPDNM, ent-6; N-methoxynonyl L-DNJ, L-MONDNJ, ent-7) ( Figure 1) were considered due to their interesting in vitro and in vivo activity toward other pathologies. 39−41 In the present study, an alternative path for the synthesis of ent-1, whose subsequent N-alkylation led to ent-(2−7), was first described. Subsequently, the capability to reduce HS accumulation and to rescue lysosomal defects by ent-(1−7) was demonstrated in a Sanfilippo B neuronal cellular model generated in our laboratory 45 and then confirmed in fibroblasts derived from Sanfilippo B patients. Furthermore, by using HeLa human epithelial cervical cancer cell line and Sanfilippo B fibroblasts, the molecular mechanism of action of the active L-iminosugars was investigated to assess whether the observed activity could be ascribed to their ability to inhibit a critical step of HS biosynthetic machinery or to increase NAGLU levels and activity.
The N-functionalization of ent-1 to obtain its N-alkyl and Nalkoxy alkyl derivatives was performed by a synthetic protocol involving the use of the well-known polymer-supported triphenylphosphine (PS-TPP)/iodine system as reported by us earlier. 40 This procedure was herein exploited to prepare ent-7, whose synthesis has never been reported before (Scheme 2). Our route involved a PS-TTP/I 2 -mediated double iodination of 1, 9nonanediol (16) to provide 1,9-diiodononane (17).
The PS-TPP/I 2 activating system (either in combination or without imidazole) has been already employed in many synthetic studies aimed to achieve different chemical transformations. 36,37,47 In this case, it can be employed in the absence of imidazole, thus allowing us to devise a synthetic procedure not requiring chromatographic purification. As a matter of fact, the resin-bound phosphine oxide, representing the sole reaction byproduct, can be simply filtered off and in this case recycled by reduction to the starting phosphine form. 48 Subsequent treatment of bis-iodide 17 with in situ-generated sodium methoxide afforded methoxy ether 18 in 75% yield. Reaction of 18 with ent-1 under standard conditions (K 2 CO 3 ) and treatment with HCl 1 M gave ent-7 as a hydrochloride salt (75% yield). Our approach enables obtaining the desired alkylated iminosugars in only a few reaction steps in satisfying yields, limiting the extractive workup and chromatographic purification stages.
Biological Evaluation. L-Iminosugars Reduce Lysosomal Defects and HS Accumulation in NAGLU-Silenced Neuroblastoma SK-NBE. In order to investigate the effect of iminosugars ent-(1−7) in cellular models of Sanfilippo disease, we used first a stable clone of the SK-NBE human neuroblastoma cell line silenced for NAGLU gene (ΔNAGLU), as a neuronal cell model of Sanfilippo B disease, which we recently generated in our lab. 45 This clone fully recapitulates the lysosomal phenotype of Sanfilippo B affected cells, thus providing a useful tool for in vitro testing the therapeutic efficacy of novel drug candidates. Indeed, silencing of NAGLU gene caused accumulation of enlarged lysosomes (Lamp1positive structures) into the cytoplasm of SK-NBE ΔNAGLU clone as compared to control clone (CTRL) stable transfected with a nontargeting shRNA ( Figure 2). SK-NBE ΔNAGLU and CTRL clones were selected to test the effect of iminosugars on the lysosomal phenotype. Both clones were grown in the presence of 20 μM of each L-iminosugar ent-(1−7) in normal growth conditions, and, after 48 h, the lysosomal accumulation was evaluated by Lamp1 staining and compared to untreated clones ( Figure 2). Treatment with any of the seven Liminosugars did not cause any change in lysosomal size and distribution of nondiseased clone (CTRL) (Figure 2a). Further assays (apoptosis and anti-proliferative effect) performed by us confirmed that ent-(1−7) did not induce any cytotoxic effect in different cell lines (IB3-1, HaCaT, and CuFi) at concentrations up to 100 μM (unpublished data). Moreover, while untreated ΔNAGLU clone showed accumulation of enlarged Lamp1 positive lysosomal structures within the cytoplasm, after treatment with L-iminosugars ent-(1−7), a significant reduction of lysosomal enlargement in ΔNAGLU clone was observed in the presence of compounds ent-1, ent-2, ent-6, and ent-7 ( Figure  2b).
No effect, instead, was highlighted with ent- (3−5). Moreover, the distribution of lysosomes inside the cytoplasm of ΔNAGLU clone treated with ent-1, ent-2, ent-6 and ent-7 appeared to be no more concentrated in the perinuclear region as they are in the untreated NAGLU-silenced clone. Indeed, as previously described by us and others, lysosomes are trapped in the perinuclear region in different lysosomal disease models. 49 In our Sanfilippo B model cell, lysosomes appeared physiologically dispersed all over the cytoplasm upon treatment with the active L-iminosugars ent-1, ent-2, ent-6, and ent-7. Physiological   To evaluate whether chirality of the iminosugar moiety exerted a key role in the observed activity, the well-known D-DNJ (1) and NBDNJ (2) were then considered. To this purpose, both CTRL and ΔNAGLU clones were grown in the presence of 20 μM of each D-iminosugar in normal growth conditions, and after 48 h the lysosomal phenotype was evaluated by Lamp1 staining as compared to untreated clones ( Figure 3). Treatment with both 1 and 2 did not cause any changes in the lysosomal size and distribution in both nondiseased model cells (CTRL) and Sanfilippo B disease model ΔNAGLU clone (Figure 3), thereby demonstrating that the efficacy highlighted by our compounds was closely related to the configuration of the pseudo-sugar moiety.
We recently demonstrated the pathological accumulation of HS on the cell membrane of SK-NBE ΔNAGLU clone and Sanfilippo B patient-derived fibroblasts as well. 45 Thus, to assess whether L-iminosugars would also be able to affect HS accumulation on the cell surface of ΔNAGLU clone, we performed indirect immunofluorescence using the anti-HS 10E4 antibody that recognizes the extracellular accumulated HS.
To this end, CTRL and ΔNAGLU clones were grown in the presence of 20 μM of each L-iminosugar ent-(1−7) in normal growth conditions, and after 48 h HS accumulation was evaluated by immunofluorescence staining (Figure 4). While CTRL clone did not show any HS accumulation (Figure 4a), the untreated ΔNAGLU clone showed consistent HS staining on cell membrane (Figure 4b). A prominent reduction of HS staining in ΔNAGLU clone was instead observed in the presence of ent-1, ent-2, ent-6, and ent-7 ( Figure 4b). Noteworthy, also in this case, ent-3, ent-4, and ent-5 were ineffective in reducing HS accumulation in ΔNAGLU clone, in line with the results already obtained with the lysosomal staining. Furthermore, treatment with any of the seven Liminosugars did not cause any change in the HS staining of the nondiseased cells (CTRL) (Figure 4a).
Overall, these results show, for the first time, that treatment with ent-1, ent-2, ent-6, and ent-7 is able to rescue both lysosomal phenotype and pathological HS accumulation on cell membrane of a cellular model of Sanfilippo B disease.
L-Iminosugars Reduce HS Accumulation and Correct Lysosomal Defects in Sanfilippo B Patient Fibroblasts. In order to test whether L-iminosugars would exert the same effects    Notably, the same compounds exerted a strong effect also on the lysosomal phenotype as highlighted above in ΔNAGLU clone. No effect, instead, was observed in the presence of iminosugars ent-3, ent-4, and ent-5 ( Figure 5).
Since we found a strong reduction of HS staining in Sanfilippo B fibroblasts, we investigated whether treatment with the same compounds would have an effect on other lysosomal diseases with HS accumulation like Sanfilippo A (MPS IIIA) and MPS I ( Figure 5). We treated fibroblasts obtained from Sanfilippo A (MPS IIIA)-and MPS I-affected patients 50 with the same compounds and dosage. Interestingly, a reduction of HS staining and lysosomal enlargement was observed by treatment with only ent-1, ent-2, ent-6, and ent-7 also in Sanfilippo A-patient-derived fibroblasts ( Figure 5). Conversely, L-iminosugars were unable to reduce HS and rescue lysosomal defects of MPS I fibroblasts, where the distribution and fluorescence intensity of lysosomes did not change as compared to untreated MPS I fibroblasts ( Figure 5). Notably, a reduction of HS staining and lysosomal defects was detectable in MPS I fibroblasts only if the concentration of ent-1, ent-2, ent-6, and ent-7 was doubled to 40 μM ( Figure 6).
These results suggest that treatment with iminosugars ent-1, ent-2, ent-6, and ent-7 is able to rescue HS and lysosome accumulation in Sanfilippo A-and B-patient-derived fibroblasts, while a higher concentration of L-iminosugars is required to affect MPS I diseased fibroblasts probably due to the accumulation of an additional GAG, the dermatan sulfate (DS), besides HS, and to the severity of the disease.
Finally, to further assess the role of iminosugar chirality, we tested the efficacy of D-enantiomers 1 and 2 in reducing lysosomal defects and HS accumulation also in patient fibroblasts. Sanfilippo A-and B-patient-derived fibroblasts, MPS I, and human adult dermal (HDFa) fibroblasts were grown on a coverslip in the presence of iminosugars 1 and 2 at the dosage of 20 μM and, after 48 h, processed for both HS and Lamp1 immunostaining. As shown in Figure 7, treatment with 1 and 2 did not have any effect on either lysosomal size and distribution or extracellular HS accumulation in both control and diseased fibroblasts.
Overall, these results indicate that, differently from 1 and 2, iminosugars ent-1, ent-2, ent-6, and ent-7, represent promising compounds worthy of further investigation in the fight against Sanfilippo B subtype and also for Sanfilippo A and MPS I.

L-Iminosugars Cause Reduction of HS Levels in Highly HS-Decorated HeLa Cells.
In order to test whether treatment with ent-(1−7) would exert the same effects on HS levels in a cell line where NAGLU gene is not mutated, we selected the HeLa human epithelial cancer cell line that is highly decorated by HS on the cell surface. Cells were grown for 48 h in the presence of ent- (1−7), and the quantity of HS was evaluated by immunostaining with the specific anti-HS antibody 10E4. 45 In accordance with all the above described results, only treatment with ent-1, ent-2, ent-6, and ent-7 caused a reduction of the cell membrane HS ( Figure S1). L-Iminosugars ent-3, ent-4, and ent-5 were also inactive in HeLa cells as in the other cellular tools used in previous experiments. Moreover, since HS is fundamental to mediate growth factor activity, we asked whether HS reduction on the cell surface would interfere with cell growth of HeLa cancer cell line. Consistent with our hypothesis, the number of HeLa cells decreased when cells were grown under treatment for 48 h with 20 μM of the active iminosugars ent-1, ent-2, ent-6, and ent-7 as compared to the untreated HeLa cells, while ent-3, ent-4, and ent-5, together with 1 and 2, did not show any significant effect on HeLa cell number count ( Figure S2a). The results herein obtained suggest that, as HeLa cells hold a nonmutated NAGLU enzyme, the HS reduction observed could be ascribed to the ability of the active iminosugars to downregulate HS synthesis. 51 Therefore, we next investigated whether treatment with the active L-iminosugars would exert their effects on HS through the reduction of protein levels of two key enzymes involved in HS synthesis, namely exostosin glycosyltransferase, EXT1, and EXT2, 52 or of the core protein of the HS proteoglycan syndecan2, SDC2. 52 We performed Western blotting analyses to evaluate protein levels of EXT1, EXT2, and SDC2 in HeLa cells untreated and treated with 20 μM of ent-1, ent-2, ent-6, and ent-7 for 24 h ( Figure S2b). The results obtained show that only ent-1 and ent-2 caused a significant decrease in protein levels of both EXT1 and EXT2 enzymes. Remarkably, treatment with ent-6 and ent-7 had a different effect on protein levels of these two enzymes, suggesting that their mechanism of action might be different from that of ent-1 and ent-2. Moreover, none of these four L-iminosugars had a relevant effect on the levels of SDC2 proteoglycan core protein.
These results suggest that the effects of ent-1 and ent-2 in reducing HS could be attributable to their ability to reduce protein levels of HS synthetic enzymes EXT1 and EXT2 in cells with a full active NAGLU enzyme.
L-Iminosugars Increase NAGLU Protein Levels and Enzymatic Activity. In order to eventually provide mechanistic  (Figure 8a, middle panel). These results suggest that the activity of these L-iminosugars in reducing HS levels in the tested cell models could be attributable to the increase in NAGLU protein levels. However, although this mechanism appears to occur in HeLa cells, this is only in part true for our Sanfilippo B model system. Treatment of SK-NBE NAGLU silenced (ΔNAGLU) clone with 20 μM of ent-1, ent-2, ent-6, and ent-7 for 24 h did not cause any increase in NAGLU protein levels (Figure 8a, lower panel), since the expression of the NAGLU interfering RNA in this clone completely abolishes NAGLU expression. Therefore, these results indicate that the mechanism of action of the selected L-iminosugars might not be related only to the increase of NAGLU protein levels. In order to evaluate the effect of the active L-iminosugars on NAGLU protein levels also in fibroblasts from Sanfilippo B patients, we treated those cells (MPS IIIB) for 24 h with 20 μM of ent-1, ent-2, ent-6, and ent-7. Remarkably, while untreated Sanfilippo B fibroblasts did not show any band for NAGLU protein, since mutated unfolded proteins are degraded by the proteasome, upon treatment with the active L-iminosugars, an increased level of NAGLU protein was detectable (Figure 8b).
In vitro enzymatic assay for the mutated NAGLU extracted from Sanfilippo B fibroblasts was then performed to establish if the iminosugars ent-1, ent-2, ent-6, and ent-7 would be able to increase NAGLU activity. To this aim, HDFa (CTRL) and Sanfilippo B extracts were used in the absence or presence of iminosugars ent-1, ent-2, ent-6, and ent-7 (20 μM) and of 4methylumbelliferyl-N-acetyl-α-D-glucosaminide as fluorogenic substrate 53,54 to measure NAGLU enzymatic activity. Normalizing for total protein concentration, HDFa showed normal NAGLU enzymatic activity, while Sanfilippo B fibroblasts had almost zero enzymatic activity as a result of the absence of NAGLU protein. The addition of ent-1, ent-2, ent-6, and ent-7 to the reaction mixture induced an up to 20 fold increase of NAGLU enzymatic activity in the case of ent-2 (Figure 8c), suggesting therefore that these active L-iminosugars might act in stabilizing the mutated enzyme.
Moreover, in order to assess if the increase of NAGLU enzymatic activity could be ascribed to the interaction of iminosugars with the enzyme active site, their inhibitory effect toward nonmutated NAGLU activity was evaluated. As a result, when ent-1, ent-2, ent-6, and ent-7 were added to the reaction mix of the HDFa lysate up to a concentration of 1 mM, no inhibition was observed, suggesting that iminosugars-NAGLU interaction involved a site different from the active one.
L-Iminosugars Rescue Proper Sorting of NAGLU Protein toward Lysosomal Compartment. We next looked at the effect of the active L-iminosugars on lysosomal sorting of NAGLU enzyme. Fibroblasts (HDFa and MPS IIIB) were incubated in the presence of 20 μM of each active L-iminosugar in normal growth conditions and, after 48 h, co-localization of NAGLU with the lysosomal associated membrane protein 1 (Lamp1) was analyzed by confocal microscopy. HDFa showed normal distribution of NAGLU enzyme within the cytoplasm colocalizing with the lysosomes, as indicated by the yellow dots. On the other hand, MPS IIIB fibroblasts showed a substantial pathological accumulation of enlarged Lamp1 positive organelles and the absence of NAGLU co-staining. Strikingly, when MPS IIIB fibroblasts were treated with ent-1, ent-2, ent-6, and ent-7, NAGLU enzyme was found to co-localize with lysosomes ( Figure 9). This result was confirmed by quantitative analysis of the total NAGLU/Lamp1 positive structures. Overall, these data further support the ability of the active iminosugars to properly sort the mutated NAGLU enzyme toward the lysosomal compartment.
L-Iminosugars Reduce Amyloid Peptide Aβ1-42 Accumulation in the Cytoplasm of ΔNAGLU Clone. Multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid β progressively aggregate in the brain of Sanfilippo patients and murine models, 55,56 resulting in a key pathogenic mechanism that contributes to the development of the neurological phenotype in Sanfilippo B patients. Therefore, we asked whether the administration of ent-1, ent-2, ent-6, and ent-7  would influence the deposition of Aβ1-42 peptide in the cytoplasm of ΔNAGLU clone. To this aim, ΔNAGLU clone was grown in the presence of 20 μM of each active L-iminosugar in normal growth conditions, and after 48 h the accumulation of Aβ1-42 peptide was evaluated by immunostaining and compared to that of the untreated clone ( Figure 10). Interestingly, for the first time, we demonstrate that in an in vitro model of Sanfilippo disease, the ΔNAGLU clone accumulates the amyloid peptide Aβ1-42 within the cytoplasm.
Moreover, treatment with the four active L-iminosugars caused a drastic reduction of the Aβ1-42 peptide accumulation in ΔNAGLU clone as compared to the untreated one ( Figure  10).
Overall, these results suggest that treatment with ent-1, ent-2, ent-6, and ent-7 could be an effective tool not only for preventing the primary cause of the disease, such as HS accumulation and lysosomal defects, but also to regulate other pathogenic mechanisms that contribute to the progression of the neurological phenotype of Sanfilippo syndrome.

■ DISCUSSION AND CONCLUSIONS
Despite clinical improvements achieved through ERT, SRT, or other therapies in patients affected by various types of LSDs, the need for new therapeutic approaches for Sanfilippo syndrome, which presents a severe neurological involvement, persists. Recently, iminosugars emerged as attractive therapeutic agents for LSDs due to their capability to penetrate the blood−brain barrier, be orally bioavailable, and have a broad tissue distribution. In the frame of our studies on the identification of the relationship between the chirality of iminosugars and their therapeutic potential, we described the activity of N-butyl-Ldeoxynojirimycin (ent-1) and some other N-alkylated Liminosugars in Pompe disease and cystic fibrosis lung disease. Herein, we demonstrated the efficacy of four synthetic iminosugars, belonging to the unnatural L-series, 40 in reducing substrate accumulation and lysosomal defects in a neuronal cell model of Sanfilippo B disease and in fibroblasts derived from Sanfilippo B-, Sanfilippo A-, and MPS I-affected patients.
Reduction of accumulated substrate in the lysosomes and/or other cellular compartments is the goal of all the developed therapies for LSDs. Indeed, substrate accumulation triggers a redistribution of the lysosomes, their expansion, and remarkable defects in their functions, including degradative activity, trafficking, and secretion. 49,57−59 In particular, an impaired lysosomal secretion, which represents a hallmark common to most LSDs, has been associated with the progressive accumulation of secondary substrates occurring in these pathologies. 26,60 Indeed, high levels of gangliosides GM2 and GM3 have been found in the brain of Sanfilippo patients, as well as in other MPS types and a variety of other LSDs. 61 Accumulation of secondary metabolites contributes to the pathophysiology of LSDs through multiple mechanisms which include impairment of vesicle trafficking and fusion of cellular membranes. 62 In addition, it is worth pointing out that in Sanfilippo syndrome and other MPS types, primary substrate accumulation is not only restricted to the lysosomes but also redistributed to various cellular and extracellular compartments. 53,63−69 Multiple evidence also highlight the role of HS localized at the cell surface or extracellularly in the progression of neurological manifestations in various MPS types, including Sanfilippo diseases, 63,70 thus paving the way to novel therapeutic approaches for these diseases. 71 In this work, we demonstrated the capability of four iminosugars, ent-1, ent-2, ent-6, and ent-7, in reducing HS accumulation in cellular models of Sanfilippo disease. Reduction of substrate accumulation in cells triggered by the active iminosugars resulted in an attenuation of the lysosomal pathologic phenotype. The activity of these compounds is strictly dependent on iminosugar chirality, as demonstrated by the observation that the iminosugars duvoglustat (1) and miglustat (2), the enantiomers of active ent-1 and ent-2, did not show any effect on HS storage and lysosomal defects in our cellular models. Thus, encouraged by these results, we performed preliminary mechanistic studies of the active iminosugars capable of restoring a normal phenotype in MPS IIIB diseased cells. In particular, from Western blotting analysis, the L-enantiomers of 1 and 2 (ent-1 and ent-2) and their analogues ent-6 and ent-7 appeared to be able to increase NAGLU protein levels (Figure 8b). Furthermore, in vitro enzymatic assays (Figure 8c) showed that the active iminosugars enhance NAGLU enzymatic activity while not acting as NAGLU inhibitors. Co-localization of NAGLU with lysosomal associated membrane protein 1 by confocal immunofluorescence microscopy revealed the ability of L-iminosugars to properly sort the mutated NAGLU enzyme toward the lysosomal compartment. These results suggest that our compounds are able to reduce the lysosomal phenotype of Sanfilippo cellular models possibly by acting as nonactive site pharmacological chaperones.
Moreover, treatment with the active L-iminosugars of epithelial cancer HeLa cells, which contain an elevated amount of HS proteoglycans on the cell surface, 72 and a nonmutated NAGLU, caused a reduction of HS, resulting in a decreased cell proliferation. These findings are consistent with the known role Table 1. Biological Activity of L-Iminosugars in Sanfilippo B Cellular Models and HeLa Cells a in ΔNAGLU (MPS IIIB) clone. b in MPS IIIB fibroblasts. c in HeLa cells.

Journal of Medicinal Chemistry
pubs.acs.org/jmc Article of HS chains as key modulators of cancer cell proliferation due to their interaction with growth factors and their receptors, leading to overstimulation of downstream signalings. 73 The active iminosugars appeared not only to act by modulating the expression and/or activity of NAGLU enzyme which catalyzes the fifth step of HS breakdown, but they also seemed to affect HS biosynthetic pathway. Our investigation demonstrated that only ent-1 and ent-2, the unnatural enantiomers of 1 and 2, respectively, reduced the expression levels of EXT1 and EXT2, while the other two active iminosugars ent-6 and ent-7 had an opposite effect. These results suggest that these compounds act through a different mechanism affecting the activity of other enzymes involved in the complex HS biosynthetic machinery that still need to be clearly understood. Indeed, EXT1 or EXT2 are members of the exostosin (EXT) family of glycosyltransferases which catalyze the elongation of HS chains through the alternate addition of glucuronic acid and N-acetylglucosamine to the HS backbone. However, HS-controlled biosynthesis involves additional enzymatic reactions (N-deacetylation and N-sulfation of glucosamine, C-5 epimerization of glucuronic acid to iduronic acid, 2-and 3-O-sulfation of uronic acid and glucosamine, respectively, and 6-O-sulfation of N-acetylated or N-sulfated glucosamine residues), as well as posttranslational modifications, which account for the enormous structural and functional heterogeneity of HS chains. 74 An overview of the results obtained in this study is reported in Table 1.
Overall, our findings enabled the identification of four Liminosugars which, after being further validated through both in vitro and in vivo tests, could contribute to the development of an effective treatment of the clinical manifestation of Sanfilippo syndrome. It is worth recalling that some of these iminosugars have been already found to hold other important pharmacological properties, and these results further augment their value in drug discovery and more generally the importance of Liminosugars, as they can now be regarded as broad-spectrum pharmacological tools rather than academic curiosities. Herein, in preliminary in vitro models, our compounds have displayed highly promising properties, demonstrating to hit the target even by more than a single mechanism of action. In addition, differently from most iminosugar drug candidates reported to date, the lack of inhibition of these L-iminosugars toward carbohydrate-processing enzymes enables association of high efficacy with selectivity and safety properties which have always limited the development of iminosugars in drug discovery. ■ EXPERIMENTAL SECTION Chemical Synthesis. General Information. All chemicals and solvents were used at the highest degree of purity and without further purification (Sigma-Aldrich, Alfa Aesar, VWR). Thin-layer chromatography analysis was carried out to follow the reaction course by using F254 Merck silica gel plates and subsequent exposure to ultraviolet radiation, iodine vapor, and spraying with ethanolic p-anisaldehyde solution. Intermediates and final products were purified by column chromatography with silica gel (70−230 mesh, Merck Kiesegel 60) and characterized by NMR analysis (NMR spectrometers: Varian Inova 500 MHz and Bruker AVANCE 400 MHz). Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis was performed with an AB SCIEX TOF/TOF 5800 MALDI mass spectrometer working in high-resolution reflectron mode. Optical rotations were measured at 25 ± 2°C in the stated solvent. Iminosugars ent-2−6 were synthesized as previously reported. 39,40 All compounds were herein converted into the corresponding hydrochloride salt by addition of 1 M HCl (1.0 equiv) followed by evaporation of volatiles. Absolute quantitative nuclear magnetic resonance (qNMR) experiments were performed to assess the purity of compounds following the "general guidelines for quantitative 1D 1 H NMR (qHNMR) experiments", provided by the Journal of Medicinal Chemistry. In all cases, purity was ≥95% (see the Supporting Information for details).
Step i: LiAlH 4 (1.69 g, 44.0 mmol) was slowly added to a stirred solution of 13 The suspension was stirred for 20 h at rt. NaOH was then added until pH = 10, and the mixture was extracted with DCM and washed with brine. The organic layers were dried (Na 2 SO 4 ), concentrated under reduced pressure, and chromatographed over silica gel (hexane/EtOAc = 6:4) to give the pure 2,3,4,6-tetra-O-benzyl-L-deoxynojirimycin (15, 8.6 g, 75% overall yield). 1 H and 13 C NMR spectra were fully in agreement with those reported in the literature for its D-enantiomer. 46 L-Deoxynojirimycin·HCl (ent-1). BCl 3 (1 M solution in DCM, 57 mL, 57.0 mmol) was added to a stirred solution of 15 (8.5 g, 16.0 mmol) in DCM (0.43 L) at 0°C. The mixture was stirred for 12 h at the same temperature and then quenched with MeOH (0.35 L) at 0°C and concentrated under reduced pressure. The crude residue was triturated with EtOAc to give pure L-DNJ·HCl (ent-1) (3.0 g, 92% yield). NMR data was consistent with the NMR spectra reported elsewhere. 39 1  1-Iodo-9-methoxynonane (18). NaH (60% dispersion in mineral oil, 0.20 g, 5.1 mmol) was added to a stirred solution of methanol (0.24 mL, 5.9 mmol) in anhydrous THF (7.5 mL) at 0°C and under an argon atmosphere. The reaction mixture was stirred at the same temperature for 1 h, then a solution of bis-iodide 17 (1.5 g, 3.9 mmol) in THF (7.5 mL) was added. The solution was warmed to rt and stirred for 48 h. Afterward, DCM was added, and the solution was washed with aq NH 4 Cl and brine. The organic layer was dried with Na 2 SO 4 and the solvent evaporated under reduced pressure. Chromatography of the crude residue over silica gel (hexane/EtOAc = 95:5) gave the pure iodide 18 (8, 0.83 g, 75% yield) as oil. 1 -7). To a stirred solution of ent-1 (0.10 g, 0.61 mmol) in anhydrous DMF (2 mL), K 2 CO 3 (0.25 g, 1.8 mmol) was added at rt under an argon atmosphere. A solution of iodide 18 (0.21 g, 0.73 mmol) in DMF (2 mL) was added dropwise and then the reaction mixture was warmed to 80°C and stirred for 16 h. The solvent was then removed under reduced pressure and chromatographed over silica gel (acetone/MeOH = 8:2) to give the pure L-MONDNJ, which was then converted into the corresponding hydrochloride salt by addition of 1 M HCl (0.61 mmol) followed by evaporation of volatiles (0.15 g, 75% yield). 1 (Taipei City,  Taiwan), and mouse anti-GAPDH monoclonal antibody (6C5 sc-32233) from Santa Cruz Biotechnology (Heidelberg, Germany) were used; secondary antibodies used were goat anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) (sc-2031) and goat anti-rabbit IgG-HRP polyclonal antibody (sc-3837) from Santa Cruz Biotechnology (Heidelberg, Germany). Cell Culture. Stable NAGLU-silenced clones were obtained from SK-NBE human neuroblastoma cell line (CRL-2271 ATCC, Wesel, Germany) as previously described. 45 SK-NBE neuroblastoma stable clones were cultured in RPMI-1640, 2 mM L-glutamine, 1 mM sodium pyruvate, supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.7 μg/mL of puromycin at 37°C in a humidified 5% CO 2 atmosphere.
Fibroblasts from MPS-affected patients were kindly provided by the Cell Line and DNA Biobank from Patients Affected by Genetic Diseases (Istituto G. Gaslini, Genoa, Italy). 50 Primary human dermal fibroblasts, adult (HDFa, from Thermo Fisher Scientific) and MPS fibroblasts were cultured in DMEM, supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, at 37°C in a humidified 5% CO 2 atmosphere.
Proliferation Assay. HeLa cells were seeded in 24-well plates at a concentration of 100,000 cells per well and grown overnight in complete RPMI medium. 76−78 HeLa cells were incubated with 20 μM of each selected L-and D-iminosugar diluted in the same medium. Control HeLa cells (mock) were untreated. After 48 h of incubation at 37°C, the cells were trypsinized, and the number of alive cells, resuspended in a solution of trypan blue, was determined by direct counts by using a BioRad automatized cell count system. The data reported are the means of three independent experiments performed with each sample in replicates of three. Error bars indicate standard errors of the means (s.e.m). Student's t-test was used for statistical comparisons, and differences were considered significant at p < 0.05. Fluorescence Microscopy. Immunofluorescence staining was performed as previously reported. 79−82 Briefly, cells grown on glass coverslips were washed with PBS and fixed in 3.7% formaldehyde at room temperature for 30 min. After fixation, cells were washed with PBS and permeabilized by incubation in blocking buffer (PBS containing 1% BSA, 0.01% sodium azide, and 0.02% saponin) for 20 min at room temperature. Cells were then incubated with the indicated primary antibodies diluted in the same blocking buffer for 1 h at room temperature. Cells were washed three times with PBS and incubated with the corresponding secondary antibodies for 30 min at room temperature. Finally, coverslips were washed in distilled water and mounted onto glass slides with the Prolong Gold anti-fade reagent with DAPI. Images were collected using a laser-scanning microscope (LSM 700, Carl Zeiss Microimaging, Inc., Jena, Germany) equipped with a Plan Apo 63× oil immersion (NA 1.4) objective lens.
Western Blotting. Cells grown to subconfluence in standard medium were harvested in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail tablet, 1 mM sodium orthovanadate, and 2.5 mM sodium pyrophosphate). 83−85 The lysates were incubated for 30 min on ice, and supernatants were collected and centrifuged for 30 min at 14,000g. Protein concentration was estimated by Bradford assay, and 50 μg/lane of total proteins were separated on SDS gels and transferred to nitrocellulose membranes. Membranes were treated with a blocking buffer (25 mM Tris, pH 7.4, 200 mM NaCl, 0.5% Triton X-100) containing 5% nonfat powdered milk for 1 h at room temperature. 86−88 Incubation with the primary antibody was carried out overnight at 4°C. After washings, membranes were incubated with the HRP-conjugated secondary antibody for 1 h at room temperature. Following further washings of the membranes, chemiluminescence was generated by an enhanced chemiluminescence (ECL) kit.
Enzyme Activity Assay. To determine NAGLU enzymatic activity in HDFa and MPS IIIB fibroblasts, pellets of 5 × 10 5 cells were collected, subjected to 10 freeze−thaw cycles, and clarified by centrifugation at 13 rpm for 30 min at 4°C. Protein concentration of samples was determined by the Lowry method. NAGLU enzymatic activity was measured as described by Marsh and Fensom using 4-methylumbelliferyl-N-acetyl-α-D-glucosaminide as the fluorogenic substrate. 54 25 μL cell lysates (HDFa and MPS IIIB) were incubated for 2 h with 50 μL of substrate solution (2 mM) and 25 μL of 0.2 M Na-acetate buffer pH 4.5. ent-1, ent-2, ent-6, and ent-7 iminosugars were added to the reaction mix of the MPS IIIB lysate at the concentration of 20 μM each. Moreover, to test the inhibitory effects of the active iminosugars toward the nonmutated NAGLU activity, ent-1, ent-2, ent-6, and ent-7 iminosugars were added to the reaction mix of the HDFa lysate at the concentration up to 1 mM. Enzymatic activity was normalized for total protein concentration, and hydrolysis of 1 nmol of substrate per hour per milligram of protein was defined as a catalytic unit.
Statistical Analysis. Data reported are expressed as the mean ± SD of at least three separate experiments. Statistical significance was determined by Student's t-test and one-way ANOVA test. ■ ASSOCIATED CONTENT
Copies of 1 H and 13 C NMR spectra and details for qNMR of synthesized compounds; additional figures concerning biological experiments (PDF) Molecular formula strings of all synthesized compounds (CSV)